A. Field of the Invention
This invention relates to novel doubIe-stranded derivatives of single-stranded DNAs derived from mungbean yellow mosaic virus (to be abbreviated as "MYMV") and to hybrid DNAs having the double-stranded derivatives inserted thereinto.
B. Description of the Prior Art
DNA genes from viruses have widely been developed and utilized as vectors in the gene recombination technology. Known viruses which give such vectors include, for example, papovaviruses such as simian virus (SV) 40 or polyoma virus, papilloma virus, and adenovirus. Since, however, these known vectors were discovered as animal vectors and do not replicate in plant cells, they cannot be utilized for gene recombination of plants.
The Ti-plasmid of an extranuclear gene possessed by Agrobacterium tumefaciens which forms tumors in dicotyledonous plants such as tomato and tobacco, and the DNA gene from cauliflower mosaic virus which causes diseases to cabbage or Chinese cabbage are the only vectors which have so far been known and have possible utilizability in plant gene recombination. No other suitable vector for plant gene recombination has yet been developed. It can be said furthermore that the above cauliflower mosaic virus is the only known plant virus having DNA genes which may possibly have utility as a vector for plant gene recombination.
Recently, Robert M Goodman et al. of University of Illinois reported that BGMV, a tropical plant virus, forms one virion from paired particles having a diameter of about 18 nm, and the genome of this virus was analyzed and found to be a circular single-stranded DNA having a size of about 2500 bases [Virology, 83, 171 (1977); Virology, 97, 388 (1979)].
Later, several kinds of plant viruses have been discovered in which paired particles having a diameter of about 18 nm form one virion and of which genes are circular and single-stranded. A group of these viruses are called "geminivirus group".
As stated above, only the cauliflower mosaic virus and geminiviruses are known as DNA-type viruses of plants, and the geminiviruses would be very promising as a goal of the development of vectors for use in plant gene recombination.
The Ti-plasmid and the cauliflower mosaic virus previously proposed in regard to vectors for use in plant gene recombination are limited to dicotyledonous plants as host plants to be infected. In contrast, the host range of the geminiviruses includes not only dicotyledonous plants but also monocotyledonous plants such as wheat and corn which are important cereals for man. Accordingly, it would seem very significant to use DNAs of these geminiviruses as vectors for plant gene recombination.
The cauliflower mosaic virus propagates in the cytoplasm of plant cells, whereas the geminiviruses do both in the cytoplasm and the nucleus. This suggests the high possibility that vectors of geminiviruses will be able to modify nuclear genes themselves of plants. If, therefore, the single-stranded DNA of the geminivirus can be used as a vector by converting it into a double-stranded DNA which is easy to handle technically in the gene recombination technology, it would be industrially valuable.
We noted that mungbean yellow mosaic virus (MYMV) is a kind of geminivirus, and made investigations on the gene of this virus. These investigations have led to the discovery that the gene of MYMV is composed of two kinds of single-stranded DNA. We have succeeded in isolating these single-stranded DNAs of MYMV. In order to use these DNAs as vectors in the gene recombination technology, we have done extensive works on the conversion of these two single-stranded DNAs into double-stranded DNAs which are technically easy to handle and on the insertion of these DNAs into other biological vectors and the propagation of the resulting hybrid DNAs in the host organisms.